Migrating cells are mechanochemical machines where biochemistry and mechanics integrate to control cellular motion. Actin polymerization driven by multiple redundant actin nucleators provides force generation for cell protrusion, an initial step of cell migration. In turn, mechanical force leads to dynamic responses of the intracellular biochemical pathways modulating actin machinery. The dissection of systems with such mechanosensitive pathways has remained a fundamental challenge in biological investigation due to the complex nature of interplay between biochemistry and mechanics in space and time.

To unravel these mechanosensitive activation patterns among multiple actin nucleators/modulators, we developed a novel statistical approach based on local image sampling and registration to directly visualize their dynamics. First, I will demonstrate how this strategy can lead to mechanistic insights into specific roles of actin nucleators using an in vitro system of actin assembly. Second, our method revealed distinct dynamics for each factor involved in cell protrusion, allowing us to establish in situ the differential functions of multiple actin nucleators/modulators. This method revealed that linear actin filaments promoted by a nucleator called formin, mDia1 initiate new protrusion, followed by exponential growth of actin branched networks by another nucleator, Arp2/3 to support edge advancement against increasing tension from the cell membrane. Finally, our quantitative method along with membrane tension perturbation showed that the dynamics of Rac1-Arp2/3 pathway is highly mechanosensitive, meaning that increasing membrane tension during protrusion is a critical factor in timely activation of Arp2/3. This suggests that migrating cells can reorganize their actin machinery in response to mechanical cues in a highly dynamic manner at a time scale of 10 s. It also highlights that mechanical processes are tightly integrated with biochemical pathways and modulate protein dynamics, thereby playing critical roles in cell physiology.